Cyanobacteria, also known as blue-green algae, are photosynthetic bacteria widespread in marine and freshwater environments worldwide. Of particular significance for water quality and human and animal health are those cyanobacteria which produce toxic compounds. A diverse range of cyanobacterial genera are well known for the formation of toxic blue-green algal blooms on water surfaces (see, for example, Codd et al., 1999; Carmichael, 2001). Many of these blooms are harmful to humans and animals due to the production of hepatotoxins and neurotoxins by the bloom-forming organisms and the ability of blooms to flourish and expand in coastal waters, streams, lakes, and in drinking water and recreational reservoirs.
There is a need for rapid methods for the accurate detection of toxic cyanobacteria to enable an assessment of the potential health hazard of cyanobacterial blooms and to allow the implementation of effective water management strategies to minimize the effects of toxic bloom outbreaks.
Two hepatotoxins of particular concern are microcystin and nodularin. Both toxins are inhibitors of eukaryotic-type protein phosphatases 1 and 2A and in vertebrates toxicity is mediated via transport of the toxins into hepatocytes. Acute exposure to either toxin can lead to liver failure and death in animals, including humans. Further, subchronic exposure to microcystin and nodularin is associated with tumor promotion and, in the case of nodularin, tumor initiation (Hitzfeld et al., 2000).
Microcystin and nodularin are cyclic peptides synthesized non-ribosomally by large multi-enzyme complexes consisting of different modules including non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) (Tillett et al., 2000; Moffitt and Neilan, 2001). These modules catalyze the activation, modification, and condensation of specific amino acids. Microcystins form a large family of cyclic heptapeptides of the general formula cyclo-(D-alanine-X-D-MeAsp-Z-Adda-D-glutamate-Mdha), where D-MeAsp is D-β-erythro-methyl-aspartatic acid, Mdha is N-methyldehydroalanine, and X and Z are variable L-amino acids. Nodularin is a cyclic pentapeptide of general formula cyclo-(D-MeAsp-L-arginine-Adda-D-glutamate-Mdhb), where Mdhb is 2-(methylamino)-2-dehydrobutyric-acid. The most unusual moiety of microcystin and nodularin is Adda (3-amino-9-methoxy-2,3,8-trimethyl-10-phenyl-4,6-decadienioc acid).
Microcystin-producing species usually produce a cocktail of different microcystin variants, however only one type will be predominately synthesized (Mikalsen et al., 2001). Only a few variants of nodularin have been identified to date.
To date there have been reports of microcystin production by cyanobacterial species from the four orders Chroococcales, Nostocales, Oscillatoriales, and Stignonematales, including Microcystis sp., Chroococcus dispersus (Chroococcales), Anabaena sp., Nostoc sp., Anabeanopsis sp. (Nostocales), Haphalosiphon, Phormidium sp., Planktothrix sp., and Oscillatoria sp. However, the genes for the production of nodularin have only been reported in N. spumigena and one N. harveyana strain (Moffitt and Neilan, 2001).
Due to this broad distribution of hepatotoxin production and sequence differences in cyanobacterial genera there is a lack of reliable molecular protocols that enable detection of all potentially hepatotoxin-producing cyanobacterial species. Most PCR-based detection methods are only based on the amplification of either microcystin or nodularin synthetase gene sequences from one genus or one species (for example Neilan, 1996; Moffitt et al., 2001; Tillet et al., 2000; Christiansen et al., 2003; Vaitomaa et al., 2003; Kurmayer et al., 2003, 2004). Protocols based on more than one data set only target the most common microcystin producing bloom-forming species Microcystis, Planktothrix, and Anabaena (Hisbergues et al., 2003). The molecular detection of other microcystin producing species such as Anabaenopsis, Phormidium, and Nostoc has not previously been addressed.
Accordingly, there is a clear need for the development of a simple detection system for the identification of multiple hepatotoxin-producing cyanobacterial species and genera.
The present inventors have now developed a molecular method that enables the detection of all known potentially microcystin and nodularin producing species with one single PCR reaction.